Isolation, Characterization, and Mapping of Four Novel Polymorphic Markers and an H3.3B Pseudogene to Chromosome 9P21-22

348J Hum Genet (1999) 44:348–349 © Jpn Soc Hum Genet and Springer-Verlag 1999

BRIEF REPORT — CASE REPORT

Elias Aliprandis · Juliette Harris · Barney Yoo Bruce D. Gelb · John A. Martignetti Isolation, characterization, and mapping of four novel polymorphic markers and an H3.3B pseudogene to chromosome 9p21-22

Received: April 16, 1999 / Accepted: May 20, 1999

Abstract Alterations in chromosomal region 9p21-22 are (Cannon-Albright et al. 1994), and multiple familial trich- among the most frequently encountered cytogenetic changes oepithelioma (Harada et al. 1996). We describe the isola- present in a number of human malignancies. In addition, the tion, characterization, fine mapping, and ordering of four causative genes of a number of hereditary cancers have been novel polymorphic markers, the previously identified genetically mapped to this region. We describe the isolation marker D9S1846, and a processed replacement histone and precise localization of four novel polymorphic markers H3.3B pseudogene and its flanking L1 and Alu elements. and a previously identified marker, D9S1846, from this region. Moreover, we have identified a retroposon-rich area within this oncogenic region containing a processed H3.3B pseudogene flanked by an L1 sequence and an Alu element. Source and isolation of polymorphic DNA markers and Together, these finely mapped and ordered reagents should an H3.3B pseudogene prove useful for genetic mapping, sequencing, and loss of heterozygosity studies of the 9p21-22 region. Two P1 clones and one PAC clone (731, 232, and 160 8P, respectively; Research Genetics, Huntsville, AL, USA) from Key words Chromosome 9p21-22 · Polymorphic markers · the region were restriction enzyme digested and the resulting Histone H3.3B Pseudogene · Loss of heterozygosity · fragments were separated electrophoretically and transferred to Retroposon a nylon membrane (NEN, Boston, MA, USA) using standard methods (Maniatis et al. 1982). The resulting Southern blot was hybridized with γ32P-ATP end-labeled di-, tri-, and tetranucleotide repeat (CA , ATA , TAG , GGAA , and Introduction n n n n GATAn) oligonucleotides. The corresponding positively hy- bridizing fragments were isolated by gel purification and Chromosomal region 9p21-22 is one of the most frequently subcloned and sequenced. Of these 12 clones, 3 were shown to deleted or translocated regions in human cancer (Mitelman be polymorphic when analyzed with a CEPH genomic DNA 1994). Malignancies associated with alterations in this re- pool (Coriell Cell Repository, Camden, NJ, USA). In order, gion include acute lymphoblastic leukemia, non-Hodgkins centromere to telomere, and based on the clone of origin and lymphoma, gliomas, pituitary adenomas, lung cancers, blad- type of repeat amplified, the markers were designated 232CA1, der tumor, ovarian cancer, and melanoma. Additionally, 160 8PGATA1, and 731CA1 (Fig. 1). three hereditary cancers have been mapped to the region: As part of the effort to identify novel genes from the 9p21- diaphyseal medullary stenosis with malignant fibrous histio- 22 region, a number of subcloned fragments from P1 clones cytoma (Martignetti et al. 1999), familial melanoma were directly sequenced. In this manner, a processed histone H3.3B pseudogene was identified within the P1 clone 526 (Fig. 2). Sequence analysis of 3.6kb revealed that the pseudogene E. Aliprandis · J. Harris · B. Yoo · B.D. Gelb · J.A. Martignetti (*) was colinear with the four exonic coding sequences of the Department of Human Genetics H3.3B gene (Albig and Doenecke 1997) including its 5Ј- and Mount Sinai School of Medicine, Box 1498, Fifth Avenue at 100th 3Ј-UTRs, the four polyA signal sites, and terminated in a Street, New York, NY 10029, USA polyA stretch. The pseudogene also possessed an additional Tel. ϩ1-212-659-6744; Fax ϩ1-212-849-2638 e-mail: [email protected] 12bp of sequence identity upstream from the reported coding start site reported by Albig et al. (1995). Alignment of the B.D. Gelb · J.A. Martignetti Department of Pediatrics, Mount Sinai School of Medicine, New sequences revealed an overall homology of 79% (1269/1613 York, USA bases). Interestingly, a partial L1 element and an Alu element 349

Fig. 2. The H3.3B pseudogene. Sequence alignment between the H3.3B (Albig et al. 1995) and H3.3Bγ3 genes was performed using the program BLAST (http://www.ncbi.nlm.nih.gov/gorf/bl2.html). The four H3.3B ex- ons are represented by open boxes and the dashed lines reveal the colinear regions between the two sequences. The relative positions of markers D9S1846 and 526GGAA1, the polyA strech, and flanking LINE 1 (oval) and Alu (arrow) sequences in the H3.3B pseudogene, and 5Ј and 3Ј UTRs Fig. 1. Chromosome placement of markers and H3.3B pseudogene in and termination signal (TGA) in the H3.3B gene are ahown the 9p21-22 region. The relative positions of the P1 and PAC clones relative to the previously ordered markers D9S162 and D9S171 are shown. Markers D9S1846 and 526GGAA1 are shown on the same line because their relative order has not been determined. The histone pseudogene is designated H3.3Bγ3 because two other H3.3B pseudogenes have been previously described (Wells and Bains 1991)

PCR conditions directly flank the H3.3B pseudogene on either end. The subse- quent insertion of these retroposons may have obscured the The PCR was performed in a volume of 20µl containing canonical flanking direct repeats that would have been gener- 10ng of genomic DNA using AmpliTaq Gold (Perkin- ated following the retroposition of the H3.3B pseudogene. Elmer, Norwalk, CT, USA) according to the manu- Inspection of the 3.6-kb sequence revealed the presence of facturer’s recommendations. Cycle conditions were 95°C two polynucleotide-rich tracts flanking the pseudo-gene (Fig. 2). for 10min, then 32 cycles of 95°C for 30s, 55°C for 30s, and Ј The 5 (CA)n dinucleotide-containing repeat segment was 72°C for 45s, with a final extension step of 10min at 72°C. shown by BLAST search (http://www.ncbi.nlm.nih.gov/ Primer pair 232CA1 was annealed at 60°C. BLAST/) to include the polymorphic microsatellite marker Ј Acknowledgments The authors thank Maria Palmer and Deirdre D9S1846. A novel 3 -tetranucleotide repeat marker, Nazareth for excellent laboratory assistance. J.A.M. was the recipient of a 526GGAA1, was shown to be polymorphic when amplified Mentored Clinical Scientist Award (NIH K08 HD 1213-02) and a Young from a CEPH DNA pool. Pediatric Investigator Award of the Mount Sinai Child Health Center supported by an NIH grant (5 P30 HD28822). This work was supported by PCR primers research grants from the American Academy of Pediatrics, Division of Human Genetics and Birth Defects; The Ruth Estrin Goldberg Grant for Cancer Research; and The Sinsheimer Scholar Award. Table 1. Primer Name Sequence Average size (bp) References 232CA1F GTG ACA GAG TGA GAC TCC 210 ATC ATA TAT Albig W, Bramlage B, Gruber K, Klobeck HG, Kunz J, Doenecke D 232CA1R GCT TGG AGG TTC AGC CAA (1995) The human replacement histone H3.3B gene (H3F3B). TAT Genomics 30:264–272 526GGAA1F TCC AGG GTG AAG ATA CAA 350 Albig W, Doenecke D (1997) The human histone gene cluster at the GTC D6S105 locus. Hum Genet 101:284–294 526GGAA1R GAC ACT ACA ATA CAT AAA Cannon-Albright LA, Goldgar DE, Neuhausen S, Gruis NA, Ander- ATG GC son DE, Lewis CM, Jost M, Tran TD (1994) Localization of the 9p 731CA1F CGT TTC ATT GCA ACC CTA 150 melanoma susceptibility locus (MLM) to a 2-cM region between ACC CT D9S736 and D9S171. Genomics 23:265–268 731CA1R CCT CTT GCT TGT AGG CCT CTC Harada H, Hashimoto K, Ko MS (1996) The gene for multiple familial TT trichoepithelioma maps to chromosome 9p21. J Invest Dermatol 107:41–43 160 8PGATA1F GCA ATG TAC AGA ACA ATG 210 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a labo- TAC ATG A ratory manual. Cold Spring Harbor Laboratory Press, Plainview, NY 160 8PGATA1R GTT TTG CTT TAT TCT TCC AGG Martignetti JA, Desnick RJ, Aliprandis E, Norton KI, Hardcastle P, ATT Nade S, Gelb BD (1999) Diaphyseal medullary stenosis with malignant fibrous histiocytoma: a hereditary bone dysplasia/cancer syn- PCR amplifications were performed using AmpliTaq Gold (Perkin- drome maps to 9p21-22. Am J Hum Genet 64:801–807 Elmer, Norwalk, CT) according to the manufacturer’s suggestions. Briefly, Mitelman F (1994) Catalog of chromosome aberrations in cancer, 5th following the initial 10 min 95°C activation step, the reaction conditions edn. Wiley/Liss, New York were [95°C for 30 sec, 56°C for 30 sec, and 72°C for 1 min] × 34 cycles then Wells D, Bains W (1991) Characterization of an unusual human his- 72 C for 10 min. Primer pair 232CA1 was annealed at 60°C tone H3.3 pseudogene. DNA Seq 2:125-127